PROJECT SUMMARY/ABSTRACT micro RNAs (miRNAs) constitute a major regulatory mechanism of gene expression. Recently, whole-genome transcriptome analyses have implicated many brain-enriched miRNAs in various mental disorders. However, our understanding of how these disease-associated miRNAs regulate cellular and signaling events in the nervous system to mediate cognitive functions is very limited. The difficulty in addressing these questions is partly due to the high degree of the heterogeneity in the cell types of the brain regions involved in the diseases and the complex and partially understood mechanisms underlying the functional readouts employed in most studies. Here, we propose to use the genetic and genomic model organism C. elegans to investigate the neuronal functions of four highly important human mental disease-related miRNAs, mir-31, mir-128, mir-134, and mir-137, by characterizing the neuronal functions of the C. elegans homologues of these miRNAs. We found that all of the C. elegans homologues of these conserved miRNAs exhibit restricted expression patterns that overlap in an interneuron RIA. RIA regulates several fundamental neural functions, including learning. We propose to use RIA as a model neuron to study the function of these well conserved miRNAs in the nervous system. First, we propose to establish the paradigm for functional characterization of these mental disease-related miRNAs using one of the conserved miRNAs, C. elegans mir-269 that is the homologue of human mir-31, as a model. An initial analysis revealed that mir-269 regulates the intracellular calcium dynamics of RIA and a form of olfactory learning, in which RIA plays a critical role. Using whole-genome transcriptome analysis of isolated RIA neurons, we identified candidate genes for the direct targets of mir-269 regulation, which includes the C. elegans homologues of the mammalian mitochondrial uniporter and its regulatory protein. Based on these results, we propose that mir-269 regulates learning by regulating mitochondria-mediated intracellular calcium dynamics of RIA. We will combine molecular genetics, optical physiology and quantitative behavioral analysis to address this hypothesis in Aim 1. Second, after establishing the system and paradigm to characterize the neuronal function of miRNAs, we will analyze the function of all of the eight C. elegans homologues of mental disease-associated miRNAs that are expressed in RIA. We will leverage our expertise in characterizing neuronal activity and behavior to provide a set of mechanistic characterization for these clinically relevant miRNAs in a defined cell type using the functional readouts that have well-characterized molecular, cellular and circuit underpinnings. Given the high degree of conservation between C. elegans and human miRNAs, our findings using this tractable model will significantly advance our understanding of the fundamental role of these mental disease-associated miRNAs, which will help to uncover the pathology of many devastating neurological diseases and to guide future therapeutical studies on these human disease conditions.